The patterns of binding of RAR, RXR and TR homo- and heterodimers to direct repeats are dictated by the binding specificites of the DNA binding domains.

We show here that, in addition to generating an increase in DNA binding efficiency, heterodimerization of retinoid X receptor (RXR) with either retinoic acid receptor (RAR) or thyroid hormone receptor (TR) alters the binding site repertoires of RAR, RXR and TR homodimers. The binding site specificities of both homo- and heterodimers appear to be largely determined by their DNA binding domains (DBDs), and are dictated by (i) homocooperative DNA binding of the RXR DBD, (ii) heterocooperative DNA binding of RXR/RAR and RXR/TR DBDs, and (iii) steric hindrance. No homodimerization domain exists in the DBDs of TR and RAR. The dimerization function which is located in the ligand binding domain further stabilizes, but in general does not change, the repertoire dictated by the corresponding DBD(s). The binding repertoire can be further modified by the actual sequence of the binding site. We also provide evidence supporting the view that the cooperative binding of the RXR/RAR and RXR/TR DBDs to directly repeated elements is anisotropic, with interactions between the dimerization interfaces occurring only with RXR bound to the 5' located motif. This polarity, which appears to be maintained in the full-length receptor heterodimers, may constitute a novel parameter in promoter-specific transactivation.     

Sustained Ca2+ signaling and delayed internalization associated with endothelin receptor heterodimers linked through a PDZ finger

G protein-coupled receptors (GPCRs), including endothelin receptor A (ETA) and B (ETB), may form dimers or higher-order oligomers that profoundly influence signaling. Here we examined a PDZ finger motif within the C-terminus of ETA and its role in heterodimerization with ETB, and in homodimerization with itself, when expressed in HEK293 cells. Receptor dimerization was monitored by (i) fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) (FRET donor) and tetracysteine/FlAsH (FRET acceptor) fused to the C-termini of ET receptors, and (ii) coimmunoprecipitation of ET receptors after mild detergent solubilization. Mutations in a PDZ finger motif at threonine403/serine404 eliminated FRET and reduced coimmunoprecipitation of heterodimers and homodimers. Functional consequences were evaluated by measuring mobilization of intracellular Ca2+ and internalization of receptors in response to a 10 nmol/L ET-1 challenge. PDZ mutations converted a sustained Ca2+ signal mediated by ETA:ETB heterodimers into a transient response, similar to that observed for homodimers or monomers. Heterodimers containing PDZ mutations were seen to internalize in a similar time domain (approximately 5 min) to the transient Ca2+ elevation and with similar kinetics to internalization of ETA homodimers or monomers. Without the PDZ mutations, heterodimers did not internalize over 15 min, suggesting the intriguing possibility that sustained Ca2+ signaling was a consequence (at least in part) of delayed internalization. The results are consistent with structural models of ETA-receptor dimerization that place threonine403/serine404 of the PDZ finger motif at the interaction interface between heterodimers and homodimers. Sustained Ca2+ signaling and delayed endocytosis of ETA:ETB heterodimers argues strongly for a unique dimer interface that impacts transmembrane signaling and internalization.     

Insight into the recognition patterns of the ErbB receptor family transmembrane domains: heterodimerization models through molecular dynamics search

ErbB receptors undergo a complex interaction network defining hierarchical and competition relationships. Dimerization is driven entirely by receptor-receptor interactions and the transmembrane domains play a role in modulating the specificity and the selection of the partners during signal transduction. To shed light on the role of the GxxxG-like dimerization motifs in the formation of ErbB transmembrane heterodimers, we propose structural models resulting from conformational search method combined with molecular dynamics simulations. Left-handed structures of the transmembrane heterodimers are found preponderant over right-handed structures. All heterotypic heterodimers undergo two modes of association either via the N-terminal motif or the C-terminal motif. The transmembrane domain of ErbB3 impairs this C-terminal motif but also associates with the other partners owing to the presence of Gly residues. The two dimerization modes involve different orientations of the two helices. Thus, a molecular-switch model allowing the transition between the two dimerizing states may apply to the heterodimers and could help interpret receptor competition for the formation of homodimers and heterodimers. The comparison between experimental and theoretical results on the dimerization hierarchy of the transmembrane domains is not straightforward. However, we demonstrate that the intrinsic properties of the transmembrane sequences are an important component in heterodimer formation and that the ErbB2 and ErbB3 transmembrane domains have a strong power for heterodimerization as observed experimentally.     

In vivo activation of PPAR target genes by RXR homodimers

The ability of a retinoid X receptor (RXR) to heterodimerize with many nuclear receptors, including LXR, PPAR, NGF1B and RAR, underscores its pivotal role within the nuclear receptor superfamily. Among these heterodimers, PPAR:RXR is considered an important signalling mediator of both PPAR ligands, such as fatty acids, and 9-cis retinoic acid (9-cis RA), an RXR ligand. In contrast, the existence of an RXR/9-cis RA signalling pathway independent of PPAR or any other dimerization partner remains disputed. Using in vivo chromatin immunoprecipitation, we now show that RXR homodimers can selectively bind to functional PPREs and induce transactivation. At the molecular level, this pathway requires stabilization of the homodimer-DNA complexes through ligand-dependent interaction with the coactivator SRC1 or TIF2. This pathway operates both in the absence and in the presence of PPAR, as assessed in cells carrying inactivating mutations in PPAR genes and in wild-type cells. In addition, this signalling pathway via PPREs is fully functional and can rescue the severe hypothermia phenotype observed in fasted PPARalpha-/- mice. These observations have important pharmacological implications for the development of new rexinoid-based treatments.     

Structural basis for retinoic X receptor repression on the tetramer

Retinoic X receptor (RXR) is a master nuclear receptor in the processes of cell development and homeostasis. Unliganded RXR exists in an autorepressed tetramer, and agonists can induce RXR dimerization and coactivator recruitment for activation. However, the molecular mechanisms involving the corepressor recruitment and antagonist-mediated repression of RXR are still elusive. Here we report the crystal structure of RXRα ligand-binding domain (LBD) complexed with silencing mediator for retinoid and thyroid hormone receptors (SMRT) corepressor motif. As the first structural report on the unliganded nuclear receptor bound to the corepressor motif, RXRαLBD-SMRT exhibits a significant structural rearrangement, compared with apoRXRαLBD tetramer. To elucidate further the molecular determinants for RXR repression by its antagonist, we also determine the crystal structure of RXRαLBD-SMRT complexed with the identified antagonist rhein. In the structure, two rhein molecules and two SMRT peptides are in the RXRαLBD tetramer, different from the case in RXRαLBD-SMRT structure, where four SMRT peptides bind to RXRαLBD tetramer. It seems that rhein induces a displacement of SMRT motif by activation function 2 (AF-2) motif binding to the receptor. Combining our current work with the published results, structural superposition of RXRαLBDs in different states reveals that RXR uses an overlapped binding site for coactivator, corepressor, and AF-2 motifs, whereas the AF-2 motif adopts different conformations for agonist or antagonist interaction and coactivator or corepressor recruitment. Taken together, we thus propose a molecular model of RXR repression on the tetramer.     

Determinants of 14-3-3σ Protein Dimerization and Function in Drug and Radiation Resistance

Many proteins exist and function as homodimers. Understanding the detailed mechanism driving the homodimerization is important and will impact future studies targeting the “undruggable” oncogenic protein dimers. In this study, we used 14-3-3σ as a model homodimeric protein and performed a systematic investigation of the potential roles of amino acid residues in the interface for homodimerization. Unlike other members of the conserved 14-3-3 protein family, 14-3-3σ prefers to form a homodimer with two subareas in the dimeric interface that has 180° symmetry. We found that both subareas of the dimeric interface are required to maintain full dimerization activity. Although the interfacial hydrophobic core residues Leu¹² and Tyr⁸⁴ play important roles in 14-3-3σ dimerization, the non-core residue Phe²⁵ appears to be more important in controlling 14-3-3σ dimerization activity. Interestingly, a similar non-core residue (Val⁸¹) is less important than Phe²⁵ in contributing to 14-3-3σ dimerization. Furthermore, dissociating dimeric 14-3-3σ into monomers by mutating the Leu¹², Phe²⁵, or Tyr⁸⁴ dimerization residue individually diminished the function of 14-3-3σ in resisting drug-induced apoptosis and in arresting cells at G₂/M phase in response to DNA-damaging treatment. Thus, dimerization appears to be required for the function of 14-3-3σ.     

Different Epidermal Growth Factor (EGF) Receptor Ligands Show Distinct Kinetics and Biased or Partial Agonism for Homodimer and Heterodimer Formation

The EGF receptor has seven different cognate ligands. Previous work has shown that these different ligands are capable of inducing different biological effects, even in the same cell. To begin to understand the molecular basis for this variation, we used luciferase fragment complementation to measure ligand-induced dimer formation and radioligand binding to study the effect of the ligands on subunit-subunit interactions in EGF receptor (EGFR) homodimers and EGFR/ErbB2 heterodimers. In luciferase fragment complementation imaging studies, amphiregulin (AREG) functioned as a partial agonist, inducing only about half as much total dimerization as the other three ligands. However, unlike the other ligands, AREG showed biphasic kinetics for dimer formation, suggesting that its path for EGF receptor activation involves binding to both monomers and preformed dimers. EGF, TGFα, and betacellulin (BTC) appear to mainly stimulate receptor activation through binding to and dimerization of receptor monomers. In radioligand binding assays, EGF and TGFα exhibited increased affinity for EGFR/ErbB2 heterodimers compared with EGFR homodimers. By contrast, BTC and AREG showed a similar affinity for both dimers. Thus, EGF and TGFα are biased agonists, whereas BTC and AREG are balanced agonists with respect to selectivity of dimer formation. These data suggest that the differences in biological response to different EGF receptor ligands may result from partial agonism for dimer formation, differences in the kinetic pathway utilized to generate activated receptor dimers, and biases in the formation of heterodimers versus homodimers.     

Microsecond time scale dynamics in the RXR DNA-binding domain from a combination of spin-echo and off-resonance rotating frame relaxation measurements

Slow protein dynamics can be studied by 15N spin-echo (CPMG) and off-resonance rotating frame relaxation through the effective field dependence of the exchange-mediated relaxation contribution. It is shown that, by a combination of these complementary techniques, a more extended sampling of the microsecond time scale processes is achieved than by either method alone. 15N R2 and improved off-resonance R1 experiments [Mulder et al. (1998) J. Magn. Reson., 131, 351–357] were applied to the 9- cis-retinoic acid receptor DNA-binding domain and allowed the identification of 14 residues exhibiting microsecond time scale dynamics. Assuming exchange between two conformational substates, average lifetimes ranging from 37 to 416 s, and chemical shift differences of up to 3 ppm were obtained. The largest perturbation of tertiary structure was observed for the second zinc finger region, which was found to be disordered in the solution structure [Holmbeck et al. (1998) J. Mol. Biol., 281, 271–284]. Since this zinc-coordinating domain comprises the principal dimerization interface for RXR in a wide repertoire of complexes with different hormone receptors to their cognate response elements, this finding has important implications for our understanding of nuclear receptor assembly on DNA direct repeats. The flexibility observed for the dimerization domain may explain how RXR, through the ability to adaptively interact with a wide variety of highly homologous partner molecules, demonstrates such a versatile DNA-binding repertoire.     

The dimerization interfaces formed between the DNA binding domains of RXR, RAR and TR determine the binding specificity and polarity of the full-length receptors to direct repeats.

Heterodimers of retinoid X receptor (RXR) and retinoic acid receptor (RAR) bind preferentially to directly repeated elements with spacing of two (DR2) or five (DR5) base pairs, due to the specific heterocooperative interaction of their DNA binding domains (DBDs) on these elements. We have demonstrated in the accompanying paper that the heterodimeric DBD interface that is responsible for the cooperative binding to DR5 elements, specifically involves the D-box of the RXR CII finger and the tip of the RAR CI finger. We show here that a second type of dimerization interface, which specifically implicates the RAR T-box and the RXR CII finger to the exclusion of the D-box, determines the selective binding to DR2 elements. Interestingly, the same type of dimerization interface (RXR T-box and CII finger) is responsible for the cooperative binding of homodimers of the RXR DBD to DR1 elements. Based on the three-dimensional structure of the glucocorticoid receptor DBD, modeling of RXR/RAR, RXR/TR and RXR/RXR DBD cooperative interactions predicts that in all cases the DBD contributing the CII finger, i.e. that of RXR, has to be positioned 5' to its cooperatively bound partner. This binding polarity of the DBDs is conferred upon the full-length receptors, since crosslinking experiments indicate that RXR is always 5' to RAR in complexes between either DR5 or DR2 and RXR/RAR heterodimers. The possible significance of these observations for transactivation by retinoic acid receptors is discussed.